Project Summary: Fragile X syndrome is a common X-linked disorder resulting in mental retardation and behavioral problems similar to autism. It is most often due to a trinucleotide repeat expansion within the FMR1 gene, which leads to transcriptional suppression and the subsequent loss of the encoded protein, FMRP. FMRP is an RNA-binding protein that, when phosphorylated and in concert with microRNAs, acts to selectively suppress translation. This grant, now in its 20 year, originally set out to clone the characteristic chromosomal fragile site associated with this disorder. This goal was met with the identification of FMR1 in 1991. In the ensuing 14 years, much has been learned about FMRP, its normal function, and the consequence of its absence. Here we propose three substantial aims to address what we consider to be three critical, but currently poorly understood, areas. First, as screening for fragile X syndrome is routinely done only by examination of the FMR1 repeat, there is a significant absence of conventional mutations. In order to define the magnitude of false negative diagnoses and to uncover experimentally useful missense mutations, we will resequence FMR1, using oligonucleotide arrays. A worldwide consortium has been established for this aim to provide appropriate patient samples. The second aim will determine the in vivo repertoire of direct RNA ligands to FMRP and its paralogs and isoforms. By immunoprecipitating cross-linked protein/RNA complexes, we will identify both mRNA and microRNA ligands. The final aim seeks mechanistic insight into FMRP-mediated translational suppression. Using mRNAs with the most robust sensitivity to FMRP, we will biochemically dissect the relationship between the mRNAs and associated microRNAs with FMRP and the process of translation. A reporter gene will then be constructed that is sensitive to FMRP activity. Such a reporter, in a neuronal cell line, will be tested as a screen for drugs that may compensate for the loss of FMRP. Relevance: The experiments proposed here are designed to address some of most contemporary questions involving fragile X syndrome. Those aspects of the proposal dealing with diagnostic issues could lead to insight having immediate affects on families touched by fragile X syndrome, while other aspects have much more long-term consequences but are designed to increase our fundamental knowledge of this disorder. Deep fundamental knowledge of a disease is the best course toward eventual therapeutics.